The invention relates to an optical zoom system for a confocal scanning microscope as well as to a confocal scanning microscope with such an optical zoom system.
Confocal scanning microscopes, which are normally constructed as laser scanning microscopes, are known in the state of the art, for example, let us cite patent DE 197 02 753 A1 in reference thereto. Most recently, components and technical systems from microscopy, specifically from confocal imaging laser scanning microscopes, have been ever more frequently applied to spectroscopic imaging techniques. In this manner, it is possible to survey the spectroscopic characteristics of a selected specimen region without destroying or touching the probed area. Confocal optic microscopy thereby makes it possible to selectively detect optical signals, which are generated within a confocal volume with limited diffraction whose magnitude lies in the realm of micrometers. Laser scanning microscopes with scanning laser beams and/or with probing feed units can generate high focal resolution for two or three dimensional representations of the specimen under examination. Owing to this characteristic, confocal laser scanning microscopy has nearly asserted itself as the standard for fluorescent probes in the field of biomedical technology.
Normally, laser scanning microscopes are used with interchangeable objectives. Thereby, the problem frequently arises that it is only with great difficulty that constant pupil positions along the optical axis can be achieved within a series of objectives. In some cases, axial differences of 40 mm can occur in the objective chamber, which can be shortened in the conjugate space of the scanning configuration between the scan mirrors by up to 4 mm. Lateral straying of the illuminating beam cone from the pupil associated with such a mismatch of the pupil's position can lead to non-uniform illumination of the specimen during scanning.
The object of the invention is therefore to create an optical configuration for a confocal scanning microscope with which the problems associated with the axially varying pupil position can be eliminated.
This task is resolved in accordance with the invention with an optical zoom system for a confocal scanning microscope, which, in the illuminating beam path of the microscope, is connected in front of the objective capturing the object, which produces an intermediate image of the object and images an entrance pupil of the illuminating beam path with variable magnification and/or with variable image length into an exit pupil.
The inventors recognized that the problems associated with the axially varying position (in the direction of illumination) of the entrance pupil of the microscope objective could surprisingly be resolved by the appropriate design of the optical zoom system. Such an optical zoom system was indeed known in the state of the art, however, from entirely different perspectives.
Laser scanning microscopes generate a specimen image in that an illuminating beam is guided over a specimen during scanning by means of a scanning arrangement and by means of a detector arrangement, which forms the image of the illuminated specimen region via the scanning arrangement by means of a confocal aperture, the irradiation originating from the illuminated spot is absorbed. The diameter of the confocal aperture determines the depth resolution and the focal resolution. The position of the confocal aperture establishes the sectional plane position in the specimen. The patent DE 196 54 211 A1 uses an optical zoom system to adjust the effective diameter of the confocal aperture or for the selection of the sectional plane position.
For laser scanning microscopes, the scanned image region can be selected by the proper control of the scanner in its zoom function, but only in the case of point to point scanning, in combination with a galvanometer scanner. In the case of parallel scanning, that is to say, scanning of several points at the same time by laser scanning microscopes, zoom function cannot be achieved by a change in the setting of the scanning arrangement since, as a rule, the individually scanned points stand in an established geometric relation to one another that is already predetermined by the configuration of the perforations in the disc, such as, for example, in the Nipkow disc, or predetermined by the aperture plate geometry in the case of a multiple pinhole aperture configuration.
The U.S. Pat. No. 6,028,306 describes such a laser scanning microscope which realizes a source for multiple spot illumination by means of a stationary confocal multiple pinhole aperture configuration, which is designed in the form of a plate with a multitude of perforations. An optical zooming unit is connected in front of the scanning arrangement, which makes it possible to magnify or scale down the multiple spot illumination. In this manner, a region of the specimen may be scanned based on a selectable size.
Such an optical zoom system known for other applications in the state of the art is used in accordance with the invention now to variably control the image length (the distance between the entrance pupil and the exit pupil on the optical zoom system), whereby fluctuations in the axial pupil position of the entrance pupil of the microscope objective can be compensated. This approach is surprising for the very reason that said construction known from the German patent DE 196 54 211 A1 does not cover the position of the pupil in the microscope as its subject matter and neither does the microscope in the U.S. Pat. No. 6,028,306. The optical zoom system in accordance with the invention therefore achieves a double function in that, on the one hand, the scanning field parameters can be adjusted by varying the magnification, and on the other hand, the transmission length can be adjusted in such a manner that an axially varying pupil position on the microscope's objective can be compensated for.
The variable magnification attained by the optical zoom system also makes it possible to change the magnification setting of the scanned field and does so specifically for multiple spot scanners operating in parallel, in which a zoom function based on intervention at the level of the scanning arrangement is not possible due to the fixed geometric interrelation of the points projected in parallel over the specimen. The known approach of controlling spot to spot scanning in confocal scanning microscopes in such a manner that an image field is scanned in the desired and adjustable magnification is just as impossible in such parallel scanning systems as it is in systems that operate with resonance scanners, that is to say, in rotating mirrors driven by resonance vibrations, since the maximum deflection available there cannot be adjusted.
A possible form of embodiment for parallel operating multiple spot scanners is represented, for example, by the known application of a Nipkow disc, as revealed in the mentioned U.S. Pat. No. 6,028,306 or in WO 8807695 or also in the European patent EP 0 539 691 A1. Beyond that, the mentioned US patent specification depicts a laser scanning microscope that scans in parallel with a multiple pinhole aperture plate which is preconnected to a corresponding microlens array such that a multiple point source is generated in the end effect. This process also lends itself for a form of embodiment of the optical zoom system. Another conceivable approach for scanning a specimen by means of parallel laser scanning microscopy, that is to say, for simultaneous scanning of multiple points, is presented by the use of a confocal slotted aperture.
The present zoom configuration is therefore particularly advantageous for application in a confocal scanning microscope which is realized with confocal multiple point imaging, in particular by means of a Nipkow disc, of a confocal slotted aperture or of a multiple point light source.
An advantageous application of an optical zoom system in accordance with the invention is furthermore provided by a confocal scanning microscope that exhibits a resonance scanner.
An objective achieves its maximum resolution in the case when the entrance pupil is fully illuminated. It is therefore efficacious to provide the appropriate means to ensure that the optical zoom system always fully illuminates the entrance pupil of the objective, regardless of the setting on the optical zoom system. As a consequence, another efficacious embodiment of the invention provides for the arrangement of an element acting as an aperture in the exit pupil of the optical zoom system, said element not being larger than the smallest exit pupil size, which occurs when the optical zoom system is in operation. As a result of this, the size of the entrance pupil is independent from the selected setting on the optical zoom system. Said size is efficaciously equal or smaller than the size the objective's entrance pupil.
During operation of the optical zoom system, the exit pupil can become very small when magnification is set to less than 1.0. If one wishes to avoid this very small exit pupil size as the lower value limit for the design, then it is efficacious to connect a telescope in front of the optical zoom system which shall affect the corresponding pupil dilation. Efficaciously, this telescope shall only be activated during beam sweep when the optical zoom system operates in the scaled down mode. In this context, the concepts of “magnify” and “scale down” here relate to the image of the specimen.
The activation of this telescope ensures that the exit pupil of the zoom, which is provided at a magnification of 1.0, can be established as the lower limit for the design without causing the exit pupil to become so small during scaled down mode of the optical zoom system that the objective's pupil might possibly become underfilled. Based in the interchangeability of the objective, it is efficacious to design the element operating as an aperture as being interchangeable if one intentionally wishes to underfill the objective's pupil, that is to say, not to fully illuminate. In that case, for example, an adjustable iris diaphragm or a mechanism with different interchangeable apertures would come under consideration such as, for example, a focal wheel with different pinhole apertures.
In an especially compactly built form of embodiment, the element acting as a lens aperture is realized by the scanning unit; for example, the limited dilatation of the scanner mirrors can act as a lens aperture.
As previously mentioned, the optical zoom system in accordance with the invention can adjust the length of the image in such a manner that an axially varying pupil position of the entrance pupil of the objective is compensated. It is therefore efficacious that the optical zoom system is controlled by a control unit to be adjustable in such a manner that in a first mode of operation, a variable image length is produced. In order to adapt the optical zoom system to an activated objective, such as to a pivoting objective, it is efficacious to maintain magnification at a constant in this mode of operation.
Once the setting for the position of the pupil is in place, another mode of operation can be advantageously realized in which the magnification is set by guidance of the control unit so as to implement a zoom function without varying the image length. By virtue of the action of the optical zoom system in this mode of operation, the scanned field can be adjusted in terms of its size. If one synchronously uses a controllable double axis scanning unit, then in addition to and depending on the adjustment change in zoom magnification, a random region can be selected within the maximum permissible scanning field as a so-called “region of interest”, whereby this “region of interest” need not be symmetrically located relative to the optical axis. During detection beam sweep, this displacement factor as well as the zoom magnification in the direction of the detector are once more cleared so that the observation of specific regions in a specimen is possible. In addition to this, images from different “regions of interest” can be acquired and subsequently recomposed into an especially highly resolved image.
An especially efficacious mode of construction of the optical zoom system uses four optical groups to implement variable pupil imaging. For the sake of manufacturing, it is favorable to provide the four optical groups, as seen in the direction of illumination, with positive refracting power, with negative refracting power as well as twice with positive refracting power. Efficaciously, at least three of the optical groups are individually and independently adjustable by means of drives, and the displacement occurs in such a manner that the focus from infinite to infinite remains intact and depending on the mode of operation, the magnification or image length (pupil position) is adjusted. It can also be advantageous to design the last group, as seen in the direction of illumination, as one unit together with a scanning objective that is standard to a confocal scanning microscope, said scanning objective being positioned in front of the scanner unit. Each group is preferably comprised of at least one lens. In order to achieve the best possible characteristics in terms of available spectral range as well as possible apertures/field angles, the groups preferably have self-correcting capabilities in terms of image defects/imaging errors.
The mentioned selection of a “region of interest” either exclusively by way of the zoom function realized by the zoom objective, or also in addition to that, by way of an asymmetrical scanning mode of operation in the possible scanned field can further be improved by the use of an element that rotates the beam path. If, for example, an Abbe König prism is inserted into the pupil of the illuminating beam path, then the scanned, zoomed scan field can be rotated. In the detection beam path (mode), this rotation is once again cleared by the prism. Such an Abbe König prism can be obtained, for example, from LINOS Photonics, Germany and is known in the state of the art. For the mentioned design, it is rotatably arranged in the beam path, in proximity of the pupil since the beam cones converge at their narrowest here, and therefore an especially small prism can be used. Depending on the rotational angle, it introduces a rotation around the double angle of the image field.